ATP-binding cassette (ABC) transporters and Niemann-pick C1 like protein (NPC1L1) play important roles in cellular sterol trafficking. ABCA1 eliminates cholesterol from cells by facilitating cholesterol efflux and the formation of nascent HDL. ABCG1 facilitates macrophage cholesterol efflux. ABCG5/ABCG8 facilitate sterol secretion into bile and sterol absorption. NPC1L1 is the putative intestinal sterol transporter. ABC transporters are gene targets of the nuclear receptors, liver X receptors (LXR). LXR are expressed in intestine, as are their ABC gene targets. This proposal will address the role of ABCA1 in intestinal HDL production; the specificity of apical sterol efflux by ABCG5/ABCG8; the role of intestinal lipid rafts in apical sterol transport; and the regulation of ABC transporters and NPC1L1 in hamster intestine by dietary cholesterol, plant sterols, or fat. Human intestinal cells, CaCo-2, will be grown on semi-permeable filters separating an upper and lower well. Regulation of ABCA1 mRNA levels and mass by dietary and biliary lipids will be studied. Lipids that alter ABCA1 expression and LXR agonists that enhance ABCA1 expression will be used to address the role of ABCA1 in intestinal HDL production. Following over expression or silencing of ABCA1 and/or ABCG1 expression, HDL production will be estimated. Human intestinal explants will be used to study the regulation of NPC1L1, ABCA1, ABCG1, ABCG5/ABCG8 mRNA, mass, and HDL production by dietary lipids and/or LXR agonists. To address the specificity of sterol uptake and efflux, expressions of ABCG5, ABCG8, or both will be over expressed or silenced. Cells will then be incubated with bile salt micelles containing labeled cholesterol, cholestenol, [unreadable]-sitosterol, or sitostanol. The rate of sterol uptake into cells, esterification, and basolateral and apical efflux will be estimated. Composition of intestinal lipid rafts will be analyzed following stimulation of apical sterol efflux or influx to ascertain whether specific proteins (caveolin-1, annexin-2, SR-B1, or others) interact to form a complex that facilitates apical sterol transport. Hamsters will be fed diets containing cholesterol or sitostanol or both, cholestyramine plus lovastatin, or different fats. The expression of ABC transporters and NPC1L1 will be estimated along the longitudinal and vertical axis of the intestine. Percent cholesterol absorption, fecal neutral sterol and bile acid excretion, and plasma and intestinal lipids will be estimated. The results of this proposal will provide new and novel information on the roles of the ABC transporters, NPC1L1, and lipid rafts in intestinal sterol metabolism [unreadable] [unreadable]